Ruminant mhc class-i-like fc receptors

ABSTRACT

Immunoglobulin G (IgG) transporting ruminant Fc receptor (FcRn) α-chain DNA molecules, especially those of cow, dromedary and sheep (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3) are disclosed. Protein expressed by said FcRn α-chain DNA molecules are disclosed and include SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6. Vectors containing the ruminant IgG transporting FcRn α-chain DNA molecules, and hosts transformed with such vectors are also included. Further, a method of producing colostrums or milk with enhanced levels of immunoglobulins or proteins fused to immunoglobulin γ-chains of FcRn interacting parts thereof is also disclosed.

The present invention relates to ruminant MHC class I-like Fc receptors,more precisely immunoglobulin G (IgG) transporting ruminant, especiallybovine (cow), dromedary and sheep, Fc receptor (FcRn) α-chain DNAmolecules, and proteins encoded by said DNA molecules. The inventionalso relates to vectors containing the DNA molecules of the invention,and hosts comprising the vectors. Additionally, the invention comprisesa method of producing milk with enhanced levels of immunoglobulins orproteins fused to immunoglobulin γ-chains or FcRn interacting partsthereof.

BACKGROUND OF THE INVENTION

The transfer of passive immunity from the mother to the calf inruminants involves passage of immunoglobulins through the colostrum (1).Upon ingestion of the colostrum, immunoglobulins are transported acrossthe intestinal barrier of the neonate into its blood. Whereas thisintestinal passage appears to be somewhat non-specific for types ofimmunoglobulins, there is a high selectivity in the passage of theseproteins from the maternal plasma across the mammary barrier into thecolostrum (2). There is a rapid drop in the concentration of all lactealimmunoglobulins immediately postpartum and the selectivity of thistransfer has led to the speculation that a specific transport mechanismacross the mammary epithelial cell barrier is involved.

The protein responsible for transfer of maternal IgG in man, mouse andrat, the FcRn, consist of a heterodimer of an integral membraneglycoprotein, similar to MHC class I α-chains, and β2-microglobulin (3).IgG has been observed in transport vesicles in neonatal rat intestinalepithelium (4). Studies have shown that FcRn is also expressed in thefetal yolk sac of rats and mice (5), in adult rat hepatocytes (6) and inthe human placenta (8, 9). More recently, Cianga et al. (9) have shownthat the receptor is localized to the epithelial cells of the acini inmammary gland of lactating mice. They have suggested that FcRn plays apossible role in regulating IgG transfer into milk in a special mannerin which FcRn recycles IgG from the mammary gland into the blood. TheFcRn is expressed in a broad range of tissues and shows differentbinding affinity to distinct isotypes of IgG and the correlation betweenserum half-life, materno-fetal transfer and affinity of different ratIgG isotypes for the mouse FcRn was recently demonstrated (10).

The present invention now provides the isolation of cDNAs encodingruminant homologues of the rat, mouse and human IgG transporting Fcreceptor, FcRn, in particular such receptors in the cow, dromedary andsheep, and their use in vectors containing the DNA molecules and hostscomprising the vectors.

SHORT DESCRIPTION OF THE INVENTION

The bovine cDNA, and deduced amino acid sequence, shows high similarityto the FcRn in other species and it consists of three extracellulardomains, a hydrophobic transmembrane region, and a cytoplasmic tail.Aligning the known FcRn sequences, we noted that the bovine proteinshows a three amino acid deletion compared to the rat and mousesequences in the α1 loop. The presence of bFcRn transcripts in multipletissues, including the mammary gland, suggests their involvement both inIgG catabolism and transcytosis. In addition, the cDNA of the fulllength coding region plus part of the 3′-end untranslated region, anddeduced amino acid sequence, of sheep, and the cDNA of dromedary missingthe first 301 nucleotides of the cDNA compare to the bovine cDNAsequence, and the deduced amino acid sequence missing the first 62 aminoacids, compared to the bovine and sheep sequences, are disclosed.

Overexpression of ruminant FcRn through transient or persistenttransgenesis using the FcRn α-chain DNA molecules according to theinvention will, either alone or by concomitant upregulation of theexpression of the corresponding β2-microglobulin gene, result in anincrease in the number of functional receptors in the udder and thusenhance the transport of immunoglobulins and/or proteins fused toimmunoglobulin γ-chains or FcRn interacting parts thereof containing theconstant region of the heavy chain of IgG. Thus, not only willantibodies acquired through natural exposure or deliberate vaccinationbe transported more effectively into the colostrum/milk, but proteinstagged with the γ-chain (i.e. proteins where the encoding gene ofinterest has been linked to sequences encoding part or the whole heavychain constant region gene for IgG), will also be more effectivelytransported into the colostrum/milk of ruminants. The latter proteinsmay be produced by animals transiently (such as through, but not limitedto DNA vaccination) or persistantly (such as through, but not limited to“conventional” transgenesis) expressing the gene construct.

The FcRn transgenic ruminant animal will express the FcRn α-chain gene(with or without concomitant β2 microglobulin expression), andexpression in the target organ can be directed by introducing thetransgene(s) directly into the udder or, through appropriate genetargeting in “conventional” transgenic animals, be expressed in theudder.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is in one aspect directed to an immunoglobulin G(IgG) transporting ruminant Fc receptor (FcRn) α-chain DNA molecule,wherein the ruminant is preferably selected from the group consisting ofcow, dromedary and sheep. In particular, the DNA molecule comprises anucleotide sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, and modified sequences of these threesequences expressing proteins with IgG transporting function.

It should be understood that the DNA molecule of the invention can beisolated and purified from biological (ruminant) sources or can beproduced by genetic engineering.

The term “modified sequences of these three sequences expressingproteins with IgG transporting function” is used in the specificationand claims to cover sequences that are truncated and sequences that havenucleotide mismatches, but still express proteins with IgG transportingfunction.

Another aspect of the invention is directed to a protein expressed by aruminant FcRn α-chain DNA molecule, wherein the ruminant is preferablyselected from the group consisting of cow, dromedary and sheep. Inparticular, the protein comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, andmodified sequences of these three sequences with IgG transportingfunction.

It should be understood that the DNA molecule of the invention can beisolated and purified from biological (ruminant) sources or can beproduced by genetic engineering.

The term “modified sequences of these three sequences with IgGtransporting function” is used in the specification and claims to coversequences that are truncated and sequences that have amino acidmismatches, but still express proteins with IgG transporting function.

Yet another aspect of the invention is directed to a vector containing aruminant IgG transporting FcRn α-chain DNA molecule according to theinvention. Examples of vectors are plasmids and phages.

Still another aspect of the invention directed to a host transformedwith a vector according to the invention. Examples of hosts arebacteria, yeasts, and ruminants, such as cows, camels, e.g. dromedaries,and sheep.

The ruminant FcRn α-chain DNA molecules of the invention and theproteins the invention may be used as tools in research work, and in theproduction of vectors of the invention.

The vectors of the invention may be used for the production of atransgenic ruminant animal or a local transgenic ruminant mother (i.e.injection into the udder).

Thus, an additional aspect of the invention is directed to a method ofproducing colostrums or milk with enhanced levels of immunoglobulins orproteins fused to immunoglobulin γ-chains or FcRn interacting partsthereof, comprising the steps of transferring a ruminant FcRn α-chainDNA molecule according to the invention through transient or persistenttransgenesis into the corresponding ruminant animal for overexpressionof a protein according to the invention, optionally at concomitantupregulation of the expression of the corresponding β2-microglobulingene, to increase the number of functional receptors in the udder,thereby enhancing the transport of immunoglobulins and/or proteins fusedto immunoglobulin γ-chains or FcRn interacting parts thereof from, orthrough, the udder into the colostrums or milk.

Examples of proteins that can be suitably produced in the milk as fusionproteins are coagulation products, such as Factor VIII, and proteinsused in medicines and food.

The invention will now be further illustrated with reference to thedescription of drawings, experiments, and sequence listing, but thescope of protection is not intended to be limited to the disclosedembodiments of the invention.

The invention is illustrated in detail with regard to the bovine (cow)FcRn gene as a representative example of a ruminant FcRn gene, but thecDNA sequence of sheep and a partial cDNA sequence of dromedary, and thecorresponding deduced amino acid sequences, are also disclosed in thesequence listing. The FcRn genes of sheep and dromedary have beenproduced by use of the same principal as used for obtaining the bovineFcRn gene. In particular, the same or similar primers have been used toamplify the FcRn alpha-chain encoding gene in sheep and dromedary.

DESCRIPTION OF THE DRAWINGS

FIG. 1. The nucleotide sequence and deduced amino acid sequence of twoforms of bovine FcRn α-chain. The potential ATG start is marked by boldcharacters, while the segment that refers to the consensus initiationsite is underlined; shaded numbers in this motif represents importantresidues (−3-A; +4-C) of the translation signal. The predictedNH₂-terminal after signal peptide cleavage is indicated by +1 under Ala.The hydrophobic membrane-spanning segment is marked by italic characterswhile the polyadenylation signal AATAAA in the 3′-UT is underlined.

The sequence data have been submitted to the NCBI Nucleotide SequenceDatabases under the accession number: AF139106.

FIG. 2. Domain by domain alignment of the predicted amino acid sequencesfor rat, mouse, bovine and human FcRn α-chains. The N-linkedglycosylation site, which is found in all the sequences is indicated bya filled triangle, while empty triangles indicate additional sites inthe rat and the mouse sequences. Dashed underline indicates residuesthat potentially interact with the Fc. The gray bar indicates thehydrophobic transmembrane region, and the asterisk represents the stopsignal in the bovine sequences. Residues in an empty box following thestop signal shows the conserved carboxyl-end of the bovine cytoplasmicdomain. Consensus residues are assigned based on the number ofoccurrences of the character in the column, emphasizing the degree ofconservation. The higher the conservation in a column the darker thebackground of the character. (Nicholas, K. B. and Nicholas, H. B. Jr.1997. GeneDoc: a tool for editing and annotating multiple sequencealignments)

FIG. 3. Scheme depicting a partial genomic DNA sequence of the bovineFcRn, which was PCR cloned applying the B7 (SEQ ID NO: 15) and B8 (SEQID NO: 16) primers. Capital letters indicate exons verified by cDNAsequence data. Exons and introns are numbered based on the genomicstructure of the mouse FcRn (19). Diagonal breaks are added wheresegments of the sequence have been deleted for reasons of space. Thedotted line indicates the splice acceptor site of intron 5, whichcarries the conserved AG dinucleotide but lacks the properpolypyrimidine tract, while the consensus splice acceptor site of intron6 is highlighted by a dashed line. The splice acceptor site of intron 5of mouse FcRn is in parenthesis under the bovine sequence indicatingsimilarities between the two segments. Underlined letters in the mousesequence indicate homology to the bovine splice acceptor site of intron5 of the bovine gene.

FIG. 4. Tissue distribution of the two forms of bovine FcRn α-chaintranscripts. A Northern blot analysis of a 1.6-kb transcript in 10 μgRNA from mammary gland (M), parotis (P), liver (L), jejunum (J), kidney(K), spleen (S) and from MDBK cell line (C) detected using a ³²P-labeledprobe from the bFcRn transmembrane-cytoplasmic region. B RT-PCR analysisof the exon 6 deleted form of bFcRn transcript. Targeted PCR for exon 6deleted cDNA amplification using B11/B12 primers (SEQ ID NO: 18 and 20,respectively).

FIG. 5. Functional expression of FcRn of different species intransfected cell lines. hFcRn/293 represents hFcRn transfected 293 cellline (7), 293 represents untransfected cells, B 1 represents bFcRntransfected rat IMCD cell lines, IMCD represents untransfected cells,rFcRn/IMCD represents rFcRn transfected IMCD cell line (14). Westernblots of total cellular protein (10 μg per lane) by using affinitypurified rabbit antisera raised against amino acids 173-187 (bovineresidues) of the α2-α3 domains.

FIG. 6. Bovine-¹²⁵I-IgG binding by bFcRn transfected IMCD cell line.Assay were done at 37° C. with (filled columns) and without (opencolumns) competing unlabeled bovine IgG, at pH 6.0 or 8.0. Each columnrepresents the mean cell-associated radioactivity in three replicates;bars show the standard error of the mean.

DESCRIPTION OF EXPERIMENTS

Materials and Methods

Cloning of a bFcRn cDNA Fragment

RT-PCR—A bovine FcRn cDNA fragment was first cloned using reversetranscription-PCR (RT-PCR). Total RNA isolated from liver by TRIzolReagent (Life Technologies, Inc., Gaithersburg, Md.) was reversetranscribed using a First-Strand cDNA Synthesis Kit (Pharmacia Biotech,Sweden). A segment spanning the α1, α2 and α3 domains was amplified bypolymerase chain reaction using two degenerate primers (B3:5′-CGCAGCARTAYCTGASCTACAA-3′ (SEQ ID NO: 7); B2:5′-GATTCCSACCACRR-GCAC-3′(SEQ ID NO: 8)) which were designed based onthe sequence homology of the published rat, mouse and human FcRnsequences (3, 5, 7).

Southern Blot Hybridization

The amplified cDNA was size fractionated on a 1-% agarose gel, blottedon a Hybond-N nylon membrane (Amersham, Arlington Heights, Ill.) andhybridized with a ³²P labeled human FcRn cDNA probe. This probe wasgenerated by RT-PCR from placental RNA using primers (HUFC2:5′-CCTGCTGGGCTGTGAACTG-3′(SEQ ID NO: 9); HUFC3:5′-ACGGAGGACTTGGCTGGAG-3′(SEQ ID NO: 10)) and encompassed a 549 bpfragment containing the α2, α3 and transmembrane regions (7). Blotscontaining the fractionated PCR amplified product of bovine cDNA washybridized in 5× Denhardt's solution, 5×SSC, 0.1% SDS, 100 μg/ml salmonsperm DNA at 60° C. for 6 hours and then washed in 2×SSC, 0.5% SDS for2×15 min at room temperature, followed by a wash in 0.1×SSC, 0.1% SDS in15 min at 60° C.

Cloning and Sequencing

Based on the expected size and Southern blot verification, the properTaq polymerase generated fragment was cloned into the pGEM-T vector(Promega Corp., Madison, Wis.). In general, preliminary sequencing wasdone by fmol DNA Sequencing System (Promega Corp., Madison, Wash.) inthe laboratory, while TaqFS dye terminator cycle sequencing wasperformed by an automated fluorescent sequencer (AB1, 373A-Stretch,Perkin Elmer) in the Cybergene company (Huddinge Sweden) to achieve thefinal sequence data

Cloning of the Full Length of bFcRn cDNA

To obtain the full length of bovine FcRn cDNA we used rapidamplification of the cDNA ends (RACE) technique (11) to isolate andclone the unknown 5′- and 3′-ends.

3′-RACE—5 μg of total RNA was reverse-transcribed by using SuperscriptII (Life Technologies, Inc., Gaithersburg, Md.) with the (dT) 17-adapterprimer (5′-GACTCGAGTCGACATCGA(T)₁₇-3′(SEQ ID NO: 11)[used also fordromedary FcRn]). The resultant cDNA was then subjected to 3′RACE-PCRamplification using the adapter primer (5′-GACTCGAGTCGACATCG-3′(SEQ IDNO: 12) [used also for dromedary FcRn]) and a bFcRn specific primer (B3(SEQ ID NO: 7)).

5′-RACE—The remaining 5′-end portion of the bovine FcRn was isolatedusing the 5′ RACE System for Rapid Amplification of cDNA Ends, Version2.0 (Life Technologies, Inc., Gaithersburg, Md.). Briefly, total RNA wasreverse transcribed using an FcRn-specific oligonucleotide (B4:5′-GGCTCCTTCCACTCCAGGTT-3′(SEQ ID NO: 13)). After first strandsynthesis, the original mRNA template was removed by treatment with theRNase mix. Unincorporated dNTPs, primer and proteins were separated fromcDNA using a GlassMax Spin Cartridge. A homopolymeric tail was thenadded to the 3′-end of the cDNA using TdT and dCTP. PCR amplificationwas accomplished using Taq polymerase, a nested FcRn-specific primer(B5: 5′-CTGCTGCGTCCACTTGATA-3′(SEQ ID NO: 14)) and adeoxyinosine-containing anchor primer. The amplified cDNA segments wereanalyzed by Southern blot analysis, cloned and sequenced as describedabove.

Cloning of a bFcRn Genomic DNA Fragment

Bovine genomic DNA was purified from liver based on the method ofAusubel (12). To analyze exon-intron boundaries of theα3-transmembrane-cytoplasmic region we PCR amplified a genomic DNAfragment using the B7 (5′-GGCGACGAGCACCACTAC-3′(SEQ ID NO: 15)) and B8(5′-GATTCCCGGAGGTCWCACA-3′(SEQ ID NO: 16)) primers. The amplified DNAwas then ligated into the pGEM-T vector (Promega Corp., Madison, Wis.)and sequenced as described above.

Tissue Distribution

Northern Hybridization

Different bovine tissue samples (mammary gland, parotis, liver, jejunum,kidney and spleen) were collected at slaughter from a lactatingHolstein-Fresian cow and frozen immediately in liquid nitrogen. Totalcellular RNA purified from these tissues and from the MDBK cell line(TRIzol Reagent, Life Technologies, Inc., Gaithersburg, Md.) (10μg/lane) was run on a denaturing agarose gel and transferred to apositively charged nylon membrane (Boehringer Mannheim GmbH, Germany).The blots were hybridized with a ³²P-labeled probe, which was generatedby Prime-A-Gene kit (Promega Corp., Madison, Wis.), containing the B7-B8(SEQ ID NO: 15-SEQ ID NO: 16) generated cDNA of the bFcRn. The finalwash was 0.1×SSC, 0.1% SDS at 60° C.

Expression and Binding Assay

The full length of bFcRn cDNA was amplified by B10(5′-CTGGGGCCGCAGA-GGGAAGG-3′(SEQ ID NO: 17) [used also for sheep FcRngene]) and B11 (5′-GAGGCAGATCACAGGAGGAGAAAT-3′(SEQ ID NO: 18) [used alsofor sheep FcRn gene]). This segment was then cloned into the pCI-neoeucaryotic expression vector (Promega Corp., Madison, Wis.). 10 μg DNAwas transfected into one 10 cm plate of IMCD cells using a CaPO₄ method(13). Cells were diluted and placed under G418 selection. IndividualG418-resistant colonies were expanded for binding assays. The presenceof the bovine FcRn in these cells was confirmed by Western blots.

Bovine IgG (Chemicon International, Temecula, Calif.) was labeled withNa¹²⁵I to a specific activity of ˜0.5 Ci/μmol using Iodogen (Pierce,Rockford, Ill.). pH dependent Fc binding and uptake was analyzedaccording to the protocol of Story et al. (7). Briefly, cells expressingthe bovine FcRn were first washed with DMEM, pH 6 or 7.5. Then,bovine-¹²⁵I-IgG in DMEM, pH 6.0 or 7.5 with or without unlabeled bovineIgG was added. The cells were allowed to bind and take up IgG for 2hours at 37° C. then unbound ligand was removed with washes of chilledPBS, pH 6.0 or 7.5. Bound radioligand was measured in a gamma counter.

Western Blot

A clone (B1) of IMCD cells transfected with cDNA encoding the bovineFcRn alpha chain, IMCD cells transfected with cDNA encoding the rat FcRnalpha chain (14), untransfected IMCD cells, 293 cells transfected withcDNA encoding the human FcRn alpha chain (7) and untransfected 293 cellswere extracted in 5% SDS. Protein extracts were resolved on gradientpolyacrylamide denaturing Tris-glycine gels (Novex, San Diego, Calif.,USA) and transferred onto PVDF (Novex). Blots were probed withaffinity-purified anti-FcRn peptide antibody, a rabbit antiserum againstthe peptide LEWKEPPSMRLKARP (SEQ ID NO: 19) representing amino acids173-187 (bovine residues) of the α2-α3 domains (14) and bound antibodywas detected with horse-radish peroxidase-conjugated goat anti-rabbitantibody and enhanced chemiluminescence (Renaissance ChemiluminescenceReagent; NEN Life Science Products Inc., Boston, Mass., USA).

Bio-Computing

Sequence comparison was completed by using the BLAST programs (15).Sequence pair distances—of bovine FcRn compare to other published FcRnsequences, was analysed by Megalign, Lasergene Biocomputing Software forthe Macintosh (DNASTAR Inc., Madison, Wis.) using the J. Hein method(16) with PAM250 residue weight table.

Results

Isolation of the Bovine FcRn cDNA

To isolate a fragment of the bovine FcRn, we first synthesized cDNA fromthe RNA isolated from bovine liver, as this tissue was previouslydemonstrated to express FcRn in other species (6, 7). PCR amplificationwith two degenerate primers (B3 and B2; SEQ ID NO: 7 and 8,respectively) yielded a DNA fragment of about 750 bp. The degenerateprimers were designed based on two conserved segments of rat (3), mouse(5) and human FcRn (7) sequences. Based on its expected size and theSouthern blot verification with a cloned human FcRn fragment, thisamplified DNA was ligated into a pGEM-T vector and one of the clones(clone 15/3) was completely sequenced. The data were compared to otherGenBank sequences using the BLAST programs, and showed high homology tothe human, rat and mouse FcRn cDNA. The insert of clone 15/3 started inthe middle of the α1 domain (exon 3) and ended in the transmembraneregion (exon 6).

We then performed 3′-RACE, using B3 (SEQ ID NO: 7) and the adapterprimer which generated a DNA fragment of ˜1.3 kbp. Several of the clonesobtained were completely sequenced. One of these (clone 4), started inthe middle of the α1 domain (exon 3) and ended with a 38-bp long poly(A)tail. The insert contained a segment of the α1, the full length of theα2, α3 domains, the transmembrane (TM) domain, the cytoplasmic (CYT)domain and ended with the 3′-untranslated (3′-UT) region. The totallength of the insert was 1304 bp excluding the poly(A)-tail. Anotherclone (clone 1) revealed complete sequence homology to clone 4 butshowed a 117 bp long deletion between the α3 domain and the cytoplasmicregion. The total length of the insert was 1187 bp excluding the poly(A)tail. The 5′ portion of the bovine FcRn was obtained by applying a5′-RACE technique. The amplification, in which we used B5 (SEQ ID NO:14) and the adapter primers, produced a 600 bp DNA fragment, which thenwas ligated into the pGEM-T vector and one of the clones (clone 5) wassequenced. The insert of clone 5 contained 567 bp, which included themissing α1, signal, and 5′-untranslated (5′-UT) regions. Clones 5 andclone 4 had an overlap of 335 bp and therefore, it was possible toobtain a composite DNA sequence of 1582 bp, encompassing the entireregion of the bovine FcRn cDNA³ (FIG. 1).

Characterization of Bovine FcRn cDNA

The sequenced and merged clones from 5′-RACE and 3′-RACE included a 116bp long 5′-untranslated region followed by an ATG initiation codon. Thismotif is flanked by nucleotides which are important in the translationalcontrol in the Kozak consensus, CC^(A)/_(G)CCAUGG, the most importantresidues being the purine in position −3 and a G nucleotide in position+4 (17). The bovine FcRn cDNA shows TCAGGATGC which is different fromthe optimal Kozak sequence. Although, bFcRn shows a purine base inposition −3 we found C instead of G in position +4 in all the clones wehave sequenced from this animal (FIG. 1). To exclude the possibility ofa Taq error during RT-PCR, we checked this motif from two other animals,and found the same sequence.

The initiation codon was followed by a 1180 bp or a 1063 bp long openreading frame in case of the full-length or the exon 6-deleted form,respectively. The exon-coded segment was followed by a 392-bp long3′-untranslated sequence including a conserved polyadenylation signal.

FIG. 2. shows the deduced amino acid sequence of the bovine FcRn (SEQ IDNO: 4) as compared to those of the human, rat and mouse. Previousstudies indicate that the structure of the characterized FcRn molecules,resembles that of the MHC class-I α-chain (3, 18). The full lengthtranscript of the bovine FcRn α-chain we isolated, is also composed ofthree extracellular domains (α1-α2-α3), a transmembrane region and acytoplasmic tail. An exon 6-deleted transcript, though, lacks theputative transmembrane region. Except for this missing domain, the twomolecules are identical at the DNA as well as at the protein level (FIG.1).

Comparing the deduced bFcRn amino acid sequence (SEQ ID NO: 4) to itshuman, rat and mouse counterparts, we found the highest overallsimilarity to the human FcRn (Table 1). Among the extracellular domains,α3-chain turned to be the most conserved, while the cytoplasmic tailreflected the highest dissimilarity. TABLE 1 Sequence pair distances (inpercent similarity) of bovine FcRn compared to published FcRn sequences,using the J. Hein method with PAM250 residue weight table α1 α2 α3 TMCYT Total Human 75.6 74.4 85.6 74.4 61.5 77.1 Mouse 61.6 66.7 78.9 66.746.2 65.9 Rat 59.3 68.9 78.9 66.7 46.2 65.4

The high similarity of the bovine FcRn as compared to the human FcRn wasfurther emphasized by analysing the end of the α1 domain. This segment,which forms a loop in the vicinity of the IgG binding site, shows a 3 ora 2 amino acid residue deletion, in the bovine and the human moleculesrespectively, compared to the rat and mouse sequences. Another commonfeature in these two molecules is that they show only one potentialN-linked glycosylation site at amino acid residue 124, based on thebovine FcRn numbering system, compared to the rat or mouse counterpartswhere there are 3 additional sites (α1-domain: position 109; α2-domain:position 150; α3-domain: position 247 based on the rat FcRn numberingsystem).

In contrast to the known FcRn sequences, we found an unusually shortcytoplasmic tail in the bFcRn where this segment is composed of 30rather than 40 amino acid residues as in all other FcRn molecules so faranalyzed. Despite its shortness, the cytoplasmic tail of the bFcRn showsthe di-leucine motif (aa: 319-320) which was previously identified as acritical signal for endocytosis but not for basolateral sorting,although, similar to the human molecule, it lacks the casein kinase II(CKII) phosphorylation site, which is found in the rat FcRn upstream ofthe di-leucine motif.

Interestingly, the nucleotides which follow the stop signal representcodons for similar amino acid residues which are found at the 3′ end ofthe human, rat and mouse molecules (FIG. 2, residues in rectangle in thebovine sequence), although it lacks the stop signal at the end of thissegment which is shared in the other FcRns. Finding this sequence in allthe clones we have analyzed and the lack of the common stop signal inthe expected conserved position, exclude the possibility of a Taq errordue to the 3′-RACE (RT-PCR) process and suggests that a mutation hasoccurred in this part of the gene.

Genomic DNA Segment of bFcRn

The two different transcripts of the bFcRn were compared to thepublished mouse genomic sequence (19). Analysis of the mouse exon-intronboundaries around α3-TM-CYT domains suggested that exon 6 is completelyeliminated from the bovine transcript representing clone 1. To verifythis hypothesis, we cloned the genomic segment of the region of interestwhich contained part of exon 5, exon 6 and a short fragment of exon 7and the two introns (intron 5 and intron 6). The B7/B8 (SEQ ID NO:15/16) amplified DNA was then cloned and sequenced. The nucleotidesequences surrounding the exon-intron boundaries revealed that thebovine splicing sites agree with their mouse counterparts (FIG. 3).Analyzing the 5′ splice site (donor site) and the 3′ splice site(acceptor site) of intron 5 and intron 6, we found that intron 5 has aconserved splice donor site (GT) while its 3′ splice site differs fromthe consensus splice acceptor sequence, which is composed of apolypyrimidine tract (PPyT) followed by an AG dinucleotide. Although theacceptor site of intron 5 has the conserved AG dinucleotide it lacks theconserved polypyrimidine tract. This non-conserved splice acceptor siteof intron 5 shows similarity to the same gene segment of the mouse FcRnsince it shows only 4 differences from the 1.5 nucleotides preceding theAG dinucleotide motif (FIG. 3). Despite this similarity, though, thereis a 14 nt long conserved PPyT in the mouse intron, followed by 24 ntand then the AG dinucleotide (19). A similar sequence was not detectedat the 3′ end of the bovine intron 5 (5′ . . . ctgtctggat ctctggtggaggactcgacc ccatccctgt cctgactcag atctgcgagg cccttaaata tctcacaacattgtctgact gcagAATCACCAGCC . . . ), whereas the splice donor and spliceacceptor sites of intron 6 shows conserved boundary sequences.

Tissue Distribution of the Two Forms of Bovine FcRn α-Chain Transcript

We then examined the tissue distribution of the two forms of the bFcRn αchain mRNA by using Northern blots and RT-PCR Based on the Northern blotanalyses, a 1.6-kb transcript was present in RNA from mammary gland,liver, jejunum, kidney, and spleen from a normal lactating HolsteinFriesian cow and the MDBK cell line (FIG. 4.A) at different levels ofexpression, whereas we did not find expression in parotis. The signalcould not represent cross-hybridization with class-I MHC mRNA since itwas detected with a probe from the transmembrane-cytoplasmic-3′-UTregion, which is dissimilar from the class I sequences. Although, thisprobe is able to detect both forms of the bFcRn, we were unable todetect the shorter transmembrane-exon-deleted form, probably because ofits low expression level or due to the low resolution of the gelelectrophoresis.

In order to analyze the expression of the alternatively spliced—exon6-deleted—transcript in tissues listed above, we performed a targetedPCR amplification (20) in which we used primer B11 (SEQ ID NO: 18) andB12 (SEQ ID NO:20). B12 corresponds to the 5′ boundary conserved regionof exon 5 juxtaposed with two conserved nucleotides in 3′ boundaryregion of exon 7. This amplification detected exon 6-deleted transcriptsin all tissues tested (FIG. 4.B).

Expression and IgG Binding of Bovine FcRn α Chain in Transfected Cells

FcRn tranfected cell lines were assessed by Western blot using rabbitantipeptide antisera raised against an epitope of human FcRn heavy chain(amino acids 174-188). Since this epitope is common in the human, in therat and in the bovine FcRn molecules, we used this antibody to detectthe expressed bovine FcRn, as well as its human and rat counterparts, ascontrols. We detected a ˜45 kDa band in the hFcRn transfected humanembryonic kidney 293 cell line, a ˜40 kDa band in the bFcRn transfectedIMCD cell lines, and two bands (˜50 kDa, and ˜55 kDa) in the rFcRntransfected IMCD cell line. The 45 kDa and the 50 kDa, 55 kDa bandsdetected of the human and rat FcRn transfected cells, are consistentwith the known molecular weight of the human and the rat FcRn α chains(6, 21), respectively. The lower band in the rat FcRn transfected IMCDcell line is the high mannose form of FcRn usually found in endoplasmicreticulum, whereas FcRn in the upper band contains complex-typeoligosaccharide chains modified in the Golgi. There was no hybridizationin the untransfected 293 and IMCD cells (FIG. 5).

The nearly 40 kDa band we detected in the bovine FcRn transfected IMCDcell line indicates that the cDNA we isolated as bovine FcRn is intactand can be fully translated. The lower moleculer weight of the bovineFcRn compare to the human and rat molecules is probably due to itsshorter cytoplasmic region and/or different glycosylation.

To determine whether the bovine FcRn clone encoded an Fc receptor, wemeasured the binding of radiolabeled bovine IgG on the bFcRn transfectedrat IMCD cell line (B1). Cells that expressed bFcRn bound IgGspecifically at pH6.0 but not at pH7.5; untransfected cells showedlittle or no specific binding at either pH (FIG. 6). A similar pHdependence of binding has previously been observed for human (7) and ratFcRn (22).

Summary of Results

The predominance of IgG1 in lacteal fluid, intestinal secretions,respiratory fluid and lacrimal fluid supports the concept of a specialrole for IgG 1 in mucosal immunity in cattle. The higher ratio ofIgG1:IgG2 in these secretions when compared to serum could represent acombination of selective IgG1 transport and local synthesis.Immunoglobulin transmission through the mammary epithelial cells is byfar the most studied, since in the cow, maternal immunity is exclusivelymediated by colostral immunoglobulins. The receptor responsible for theIgG transport has not been identified prior to the present invention,although previous studies have indicated that specific binding sitesexist on bovine mammary epithelial cells near parturition which arepresumably involved in the transfer of IgG1. We have now isolated andcharacterized a cDNA encoding a bovine homologue of the human, rat andmouse IgG transporting Fc receptor, FcRn.

Sequence Analysis

Extracellular Backbone and the FcRn/Fc Interaction Site

The bovine cDNA and its deduced amino acid sequence were similar to therat, mouse and human FcRns (FIG. 2) (3, 5, 7). Among these sequences,the bovine α chain shows the highest overall similarity to its humancounterpart (Table 1).

Based on the crystal structure of a 2:1 complex of FcRn and the Fcfragment of rat IgG (18) the approximate binding region on each moleculecould be localized. The first contact zone of the heavy chain of the ratFcRn molecule can be found at the end of the α1 domain involvingresidues 84-86, and 90. The second contact zone involves residues113-119, while the third contact zone encompasses residues 131-137, bothsegments are part of the α2 domain.

The close relationship between the human and bovine FcRn molecules wasfurther emphasized by analyzing the end of the α1 domain, which wassuspected to form the first contact zone in the rat FcRn/Fc interaction.Both the bovine and human FcRns are three and two amino acid residuesshorter, respectively, compared to their rodent counterparts. It isinteresting that these deletions eliminate an N-linked glycosylationsite found in their rat and mouse counterparts and which is ubiquitousin MHC class-I proteins.

The second contact zone, which is part of the α2 domain, is wellconserved, emphasizing its importance in IgG binding. Another differenceof the bovine FcRn compared to the rat molecule is a radical amino acidsubstitution at the third contact zone (aa: 134-Arg) in the α2 domain.These observations suggest critical importance of the second and thirdcontact zones, while those residues that make up the first contact zoneare probably less crucial in the IgG/FcRn interaction in the cow andalso in humans, further supporting the conclusion of Vaughn et al. (24)who applied site directed mutagenesis to analyze the role of thepredicted contact residues of the rat FcRn. They found that replacementof residues 84-86 of the α1 domain, which was thought to be the firstcontact zone, did not significantly alter binding affinity.

We found that the critical residues of the α3 domains (aa: 216L, 242K,248H, 249H), which also influence the FcRn/Fc interaction are conservedamong the different species thus far analyzed. The bFcRn, similarly toits human counterpart, has an absence of the N-linked glycosylation sitein the α3 domain, which is of interest, since for rat FcRn this has beensuggested to mediate FcRn dimerization via a carbohydrate handshake(22).

In this context one might predict that in the cow, the mammaryepithelial cells are able to carry IgG via FcRn mediated transcytosisfrom the blood into their secretory fluid, although none of the studiesindicated pH dependent IgG binding, which we found in analyzing IgGbinding to the bovine FcRn (FIG. 6).

In summary, our data indicate that the FcRn transcripts are expressed indifferent tissues, including the mammary gland, in cattle, andstrengthens their suggested involvement in IgG catabolism andtranscytosis (for review see Junghans, 1997 (23)). It will be ofinterest to investigate the bFcRn binding affinity or the transportefficiency mediated by this receptor of the bovine IgG subclasses.Analyses of the localization and the expressional level of the bFcRn inthe mammary gland at different times during the lactation period mayalso help to clarify its function in the transport of IgG into thecolostrum.

Production of Proteins Fused to Immunoglobulin γ-Chains

Examples of techniques of producing proteins fused to immunoglobulinγ-chains are described in a number of publications (e.g. 24-35) and willtherefore not be described herein.

Production of Transgenic Ruminants

Examples of techniques of producing transgenic animals are disclosed inmany prior art publications (e.g. transgenic sheep (36-52) andtransgenic cows (53-67)) and will not be described herein.

However, the teachings of all references cited in the presentspecification are hereby included by reference.

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1. A method of producing colostrums or milk with enhanced levels ofimmunoglobulins or proteins fused to immunoglobulin γ-chains or FcRninteracting parts thereof, comprising the steps of transferring animmunoglobulin G (IgG) transporting ruminant Fc receptor (FcRn) α-chainDNA molecule through transient or persistent transgenesis into thecorresponding ruminant animal for overexpression of the proteinexpressed by the ruminant FcRn α-chain DNA molecule, optionally atconcomitant upregulation of the expression of the correspondingβ2-microglobulin gene, to increase the number of functional receptors inthe udder, thereby enhancing the transport of immunoglobulins and/orproteins fused to immunoglobulin γ-chains or FcRn interacting partsthereof from, or through, the udder into the colostrums or milk.
 2. Themethod according to claim 1, wherein the ruminant of the immunoglobulinG (IgG) transporting ruminant Fc receptor (FcRn) α-chain DNA molecule isselected from the group consisting of cow, dromedary and sheep.
 3. Themethod according to claim 2, wherein the DNA molecule has a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, and modified sequences of these three sequencesexpressing proteins with IgG transporting function.
 4. The methodaccording to claim 1, wherein the ruminant of the protein expressed bythe ruminant FcRn α-chain DNA molecule is selected from the groupconsisting of cow, dromedary and sheep.
 5. The method according to claim4, wherein the protein has an amino acid sequence selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, andmodified sequences of these three sequences with IgG transportingfunction.